microRNA-29a Regulates ADAM12 Through Direct Interaction With ADAM12 mRNA and Modulates Postischemic Perfusion Recovery.

Background Peripheral artery disease is caused by atherosclerotic occlusion of vessels outside the heart and most commonly affects vessels of the lower extremities. Angiogenesis is a part of the postischemic adaptation involved in restoring blood flow in peripheral artery disease. Previously, in a murine hind limb ischemia model of peripheral artery disease, we identified ADAM12 (a disintegrin and metalloproteinase gene 12) as a key genetic modifier of postischemic perfusion recovery. However, less is known about ADAM12 regulation in ischemia. MicroRNAs are a class of small, noncoding, single-stranded RNAs that regulate gene expression primarily through transcriptional repression of messenger RNA (mRNA). We showed microRNA-29a (miR-29a) modulates ADAM12 expression in the setting of diabetes and ischemia. However, how miR-29a modulates ADAM12 is not known. Moreover, the physiological effects of miR-29a modulation in a nondiabetic setting is not known. Methods and Results We overexpressed or inhibited miR-29a in ischemic mouse gastrocnemius and tibialis anterior muscles, and quantified the effect on perfusion recovery, ADAM12 expression, angiogenesis, and skeletal muscle regeneration. In addition, using RNA immunoprecipitation-based anti-miR competitive assay, we investigated the interaction of miR-29a and ADAM12 mRNA in mouse microvascular endothelial cell, skeletal muscle, and human endothelial cell lysates. Ectopic expression of miR-29a in ischemic mouse hind limbs decreased ADAM12 mRNA expression, increased skeletal muscle injury, decreased skeletal muscle function, and decreased angiogenesis and perfusion recovery, with no effect on skeletal muscle regeneration and myofiber cross-sectional area following hind limb ischemia. RNA immunoprecipitation-based anti-miR competitive assay studies showed miR-29a antagomir displaced miR-29a and ADAM12 mRNA from the AGO-2 (Argonaut-2) complex in a dose dependent manner. Conclusions Taken together, the data show miR-29a suppresses ADAM12 expression by directly binding to its mRNA, resulting in impaired skeletal muscle function, angiogenesis, and poor perfusion. Hence, elevated levels of miR-29a, as seen in diabetes and aging, likely contribute to vascular pathology, and modulation of miR-29a could be a therapeutic target.

MicroRNA regulation of BAG3.

B-cell lymphoma 2 (Bcl-2)-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that modulates major biological processes, including apoptosis, autophagy, and development to promote cellular adaptive responses to stress stimuli. Although BAG3 is constitutively expressed in several cell types, its expression is also inducible and is regulated by microRNAs (miRNAs). miRNAs are small non-coding RNAs that mostly bind to the 3'-UTR (untranslated region) of mRNAs to inhibit their translation or to promote their degradation. miRNAs can potentially regulate over 50% of the protein-coding genes in a cell and therefore are involved in the regulation of all major functions, including cell differentiation, growth, proliferation, apoptosis, and autophagy. Dysregulation of miRNA expression is associated with pathogenesis of numerous diseases, including peripheral artery disease (PAD). BAG3 plays a critical role in regulating the response of skeletal muscle cells to ischemia by its ability to regulate autophagy. However, the biological role of miRNAs in the regulation of BAG3 in biological processes has only been elucidated recently. In this review, we discuss how miRNA may play a key role in regulating BAG3 expression under normal and pathological conditions.

Modulation of miR-29a and ADAM12 Reduces Post-Ischemic Skeletal Muscle Injury and Improves Perfusion Recovery and Skeletal Muscle Function in a Mouse Model of Type 2 Diabetes and Peripheral Artery Disease.

Both Type 1 diabetes mellitus (DM1) and type 2 diabetes mellitus (DM2) are associated with an increased risk of limb amputation in peripheral arterial disease (PAD). How diabetes contributes to poor PAD outcomes is poorly understood but may occur through different mechanisms in DM1 and DM2. Previously, we identified a disintegrin and metalloproteinase gene 12 (ADAM12) as a key genetic modifier of post-ischemic perfusion recovery. In an experimental PAD, we showed that ADAM12 is regulated by miR-29a and this regulation is impaired in ischemic endothelial cells in DM1, contributing to poor perfusion recovery. Here we investigated whether miR-29a regulation of ADAM12 is altered in experimental PAD in the setting of DM2. We also explored whether modulation of miR-29a and ADAM12 expression can improve perfusion recovery and limb function in mice with DM2. Our result showed that in the ischemic limb of mice with DM2, miR-29a expression is poorly downregulated and ADAM12 upregulation is impaired. Inhibition of miR-29a and overexpression of ADAM12 improved perfusion recovery, reduced skeletal muscle injury, improved muscle function, and increased cleaved Tie 2 and AKT phosphorylation. Thus, inhibition of miR-29a and or augmentation of ADAM12 improves experimental PAD outcomes in DM2 likely through modulation of Tie 2 and AKT signalling.